How to convert ELISA format assays to xMAP using MagPlex beads for MAGPIX Purpose This publication will provide guidance on how to create a microsphere-based assay utilizing antibody pairs that you are currently using in an ELISA or a new set of antibodies. With your xMAP format assay, you will decrease spending on antibodies, use less sample, have the option of utilizing automation, and see increased sensitivity and dynamic range. The вЂ�openвЂ™ platform of xMAP allows for rapid addition to an already existing single-plex or multiplex panel (Faucher et al. 2009; Kofoed et al. 2006). This publication specifically addresses how to create a washed sandwich immunoassay using magnetic beads. If you are interested in building a different type of assay such as a no-wash, competitive, serology, etc., please see the Luminex FAQ page, http://www.luminexcorp.com/Support/SupportResources/index.htm. The references at the end of this document have all utilized xMAP for creation of sandwich immunoassays to replace ELISAs. A number of publications built assays for human cytokines, and others have developed assays for porcine (Bjerre et al. 2009, Johannison et al. 2006, Lawson et al. 2010), bovine (Dernfalk et al. 2004, Dernfalk et al. 2007), equine (Wagner et al. 2009 and 2010), canine (Wagner et al. 2011) and feline (Wood et al. 2011) cytokines as well as plant proteins (Haasnoot et al. 2007). Assays have been built as just a single-plex due to the advantages previously stated, such as reduced sample volume used and increased sensitivity (Faucher et al. 2009; Rizzi et al. 2010). Contents Purpose ....................................................................................................................................... 1 Contents ...................................................................................................................................... 1 Introduction .................................................................................................................................. 2 Selection of Assay Components ................................................................................................. 2 Bead Coupling ............................................................................................................................. 3 Recommended Protocol for Two-Step Carbodiimide Coupling of Antibodies to MagPlexпЈЁ Magnetic Carboxylated Microspheres .................................................................................... 5 Confirmation of Coupling ........................................................................................................... 10 Recommended Protocol for Confirmation of Antibody Coupling .......................................... 14 Selecting the best Pairs of Antibodies....................................................................................... 16 Assay Protocol/Performing the Assay ....................................................................................... 16 В® Recommended Protocol for Washed Capture Sandwich Immunoassay Using MagPlex Microspheres ........................................................................................................................ 18 References ................................................................................................................................ 22 Appendix вЂ“ Automated Washing Option Using Bio-Tek ELx405 Microplate Washer ............... 25 3/15/2011 ADV-002 Rev A 1 Introduction For the last four decades, enzyme-linked immunosorbent assays (ELISA) developed by Engvall and Perlmann (1971) have been the primary source for detection of protein biomarkers in biological samples for both life science research and clinical diagnostics. While widely utilized (Lequin, 2005), ELISA has limitations. An ELISA is typically performed in a 96-well microplate and the wells are coated with relatively large quantities of capture antibody. Also, in order to capture antigen of interest from the sample, large sample volumes are required. xMAP format assays have the added value of higher throughput, increased flexibility, reduced sample volume, and lower costs with a similar workflow as ELISA. LuminexвЂ™s xMAP Technology has been utilized in over 8,000 publications (www.luminexcorp.com/bibliography), and can be applied to both protein and nucleic acid applications. The suspended beads allow for assay flexibility in a single-plex or multiplex format. Since the beads have the capture antibody immobilized on their much smaller surface area as compared to a microplate well, smaller sample volumes are required and non-specific binding is reduced (Carson & Vignali 2000). Recent enhancements by Luminex include the new MAGPIX system and the introduction of magnetic microspheres which have reduced the cost of performing a single-plex assay as expensive filter microplates are no longer required. The magnetic beads also make the workflow easier and are automation compatible. Selection of Assay Components Manufacturers of ELISA kits often sell matched pairs that are easily transferable to beads. Examples include but are not limited to, DuoSetsВ® from R&D Systems (Wood et al. 2011) and eBioscience (Rizzi et al. 2010). Many publications list the source, catalog number and clone number for their antibodies used (Bjerre et al. 2009; Carslon & Vignali 1999; de Jager et al. 2003; de Jager et al. 2005; de Jager et al. 2009; Dernfalk, et al. 2004; Dernfalk et al. 2007; Lawson et al. 2010; Ray et al. 2005; Skogstrand et al. 2005). Many of the assays built in these publications are common and Luminex suggests you use these as a starting point to save time. The Antibody Resource website (http://www.antibodyresource.com) is a good starting point to search for antibody suppliers. When choosing raw materials (antibodies and recombinant proteins), select vendors that have rigorous quality control procedures and provide as much information as possible about the antibodies or proteins. Request that the vendor provide purity information from SDS- and nondenaturing-PAGE. Also, request profiles of the antibody from capillary isoelectric focusing to compare lots from the same vendor. DonвЂ™t rely upon the vendor to quality control raw materials for you. Luminex recommends that you devise your own incoming materials quality control procedure. When coupling antibodies to the beads itвЂ™s ideal to use a monoclonal antibody as the capture. A polyclonal or monoclonal can be used as the detection antibody. Monoclonal antibodies used for capture should be specific for a different epitope than the detection antibody. Couple every antibody to the microspheres. Try to find antibodies that come in both plain and biotinylated versions. If you need to biotinylate antibodies there is an example in Lawson et al. 2010. Also, Pierce has a product, EX-Link Sulfo-NHS-LC-Biotin catalog #21335, for this purpose. 3/15/2011 ADV-002 Rev A 2 Bead Coupling Luminex MagPlex superparamagnetic microspheres are covered with approximately 10 million carboxyl groups for binding proteins or other macromolecules. Proteins are coupled to the beads by conjugating the free amines of lysine sidechains and the N-terminal amines of proteins to the carboxyl groups on the beads. Bead coupling is a two-step procedure. The first step is to activate the microspheres with EDC/Sulfo-NHS. The excess is removed and then protein is added. After the protein is conjugated to the beads, the remaining sites are blocked with detergent or BSA. Proteins conjugate best when maintained in the storage buffer the manufacturer provided but if interfering substances are present, buffer exchange into an alternative buffer of the same pH is recommended. Table 1. Suggested Mag-Plex bead regions to utilize for building an immunoassay Region Part # Region Part # Region Part # Region Part # Region Part # 12 MC10012 26 MC10026 38 MC10038 52 MC10052 65 MC10065 13 MC10013 27 MC10027 39 MC10039 53 MC10053 66 MC10066 14 MC10014 28 MC10028 42 MC10042 54 MC10054 67 MC10067 15 MC10015 29 MC10029 43 MC10043 55 MC10055 72 MC10072 18 MC10018 30 MC10030 44 MC10030 56 MC10056 73 MC10073 19 MC10019 33 MC10033 45 MC10045 57 MC10057 74 MC10074 20 MC10020 34 MC10034 46 MC10046 61 MC10061 75 MC10075 21 MC10021 35 MC10035 47 MC10047 62 MC10062 76 MC10076 22 MC10022 36 MC10036 48 MC10048 63 MC10063 77 MC10077 25 MC10025 37 MC10037 51 MC10051 64 MC10064 78 MC10078 6 Note: Bead regions in bold are also available in lower concentrations of 2.5 x 10 beads/ml. Part Number Format: MC100XX-ID (where XX is the region) 3/15/2011 ADV-002 Rev A 3 Typically, Luminex suggests coupling approximately 5 Вµg of antibody per 1 million beads as a starting point. If optimization is required, try coupling in the 1 to 25 Вµg per 1 million bead range. Microsphere coupling reactions can be easily scaled up or down depending upon user needs. Please refer to Bjerre et al. (2009) for an example of coupling titration results and Table 2 for bead and reagent information. Here are some things to keep in mind for coupling reactions: вЂў Do not couple less than one million microspheres at a time вЂў Mixing volume is very important for protein coupling and is based on the number of beads you are coupling and the size of the vessel you are using. Table 2. Scale-up Information for Bead Coupling EDC SulfoNHS Activation volume 0.5mg 0.5mg 0.5mg 2.5mg 2.5mg 2.5mg 5mg 5mg 100Вµl 100Вµl 100Вµl 100Вµl 100Вµl 500Вµl 500Вµl 500Вµl Microspheres 1 million 2.5 million 5 million 10 million 12.5 million* 50 million 100 million 200 million 0.5mg 0.5mg 0.5mg 2.5mg 2.5mg 2.5mg 5mg 5mg Coupling Volume S-NHS (50mg/ml) EDC (5mg/ml) 0.5mL 0.5mL 0.5mL 1mL 1mL 2mL 2mL 2mL 10ul 10ul 10ul 10ul 10ul 10ul 100ul 100ul 10ul 10ul 10ul 10ul 10ul 10ul 100ul 100ul ~ # of 96well plates using 2500 beads/well 4 10 20 40 50 200 400 800 *Standard 1ml vials of microspheres contain 12.5 million microspheres Coupling reactions of 0.5 to 1ml are performed in 1.5 mL microcentrifuge tubes, and 2ml coupling reactions are performed in 4mL microcentrifuge tubes or 15ml polypropylene centrifuge tubes. The stability of your coupled beads will depend upon the antibody coupled to the beads. Antibody coupled microspheres are stable for approximately one year at 4В°C storage. Important notes about EDC: . вЂў EDC and Sulfo-NHS are both water sensitive and once resuspended can only be used once вЂў Store EDC and Sulfo-NHS at -20 C in a dessicator вЂў Aliquot EDC into single-use portions as it is hygroscopic and will degrade if opened multiple times. Alternatively, convenient single-use 10 mg vials are commercially available. 3/15/2011 ADV-002 Rev A 4 Recommended Protocol for Two-Step Carbodiimide Coupling of Antibodies to MagPlexпЈЁ пЈЁ Magnetic Carboxylated Microspheres Microspheres should be protected from prolonged exposure to light throughout this procedure. Table 3. Required Materials for Bead Coupling Source Catalog Number MagPlex Microspheres Capture Antibody Luminex Various Vortex Mixer Sonicator (mini) Pipettors P10, P20, P100, P1000, 8 ch. Hemacytometer (Bright Line) OR automated cell/particle counter Rotator 1.5 mL co-polymer microcentrifuge tubes OR 1.5 mL Eppendorf Protein LoBind microcentrifuge tubes 2.0 mL screw-cap microcentrifuge tubes for storage 10 ВµL pipette tip refills 250 ВµL pipette tip refills 1000 ВµL pipette tip refills Dynal MPCВ®-S Magnetic Particle Separator EDC (1-ethyl-3[3dimethylaminopropyl] carbodiimide hydrochloride) Sulfo-NHS (Nhydroxysulfosuccinimide) VWR Cole Parmer Rainin or other Refer to Table 1 Various (see references for examples) 58816-121 08849-00 Various VWR 15170-172 VWR USA Scientific 56264-302 1415-2500 Eppendorf Fisher Fisher 022431081 13-698-794 05-669-8 Rainin Rainin Rainin Invitrogen GPS-10G GPS-250 GPS-1000 120-20D Pierce 77149 or 22980 Pierce 24510 Product 3/15/2011 ADV-002 Rev A 5 Table 4. Recipe for 0.1 M NaH2PO4, pH 6.2 (Activation Buffer)* 250 mL Reagent Catalog Number Sigma S3139 Final Concentration 0.1 M NaH2PO4 Amount/ 250 mL 3g NaH2PO4 (Sodium phosphate monobasic, anhydrous) 5 N NaOH Fisher SS256-500 ------ ~ 40 drops Filter Sterilize and store at 4В°В°C * Activation can be performed in 50 mM MES, pH 6.0-6.2 with similar results. Table 5. Recipe for 0.05 M MES, pH 5.0 (Coupling Buffer)** 250 mL Reagent Catalog Number MES (2[N-Morpholino] ethanesulfonic acid) 5 N NaOH Sigma M2933 Fisher SS256500 Final Concentration 0.05 M MES Amount/ 250 mL 2.44 g ------ ~ 5 drops Filter Sterilize and store at 4В°C ** Coupling can be performed in 100 mM MES, pH 6.0 with similar results. For some proteins, better solubility and better coupling may be achieved at a higher coupling pH. Table 6. Recipe for PBS, 0.1% BSA, 0.02% TWEEN-20, 0.05% Azide, pH 7.4 (PBS-TBN Blocking/Storage Buffer) 1000 mL Reagent PBS, pH 7.4 Catalog Number Sigma P3813 BSA TWEENВ® 20 Sigma A7888 Sigma P9416 Sodium Azide Sigma S8032 Final Concentration 138 mM NaCl 2.7 mM KCl 0.1% BSA 0.02% TWEENВ® 20 0.05% Azide Amount/ 1L 1 packet 1g 0.2 mL 500 mg Filter Sterilize and store at 4В°C 3/15/2011 ADV-002 Rev A 6 1. Resuspend the stock uncoupled microsphere suspension according to the instructions described in the Product Information Sheet provided with your microspheres. 2. Transfer 5.0 x 106 of the stock microspheres to a USA Scientific microcentrifuge tube. 3. Place the tube into a magnetic separator and allow separation to occur for 60 to 120 seconds. 4. With the tube still positioned in the magnetic separator, remove the supernatant. Take care not to disturb the microspheres. 5. Remove the tube from the magnetic separator and resuspend the microspheres in 100 ВµL dH2O by vortex and sonication for approximately 20 seconds. 6. Place the tube into a magnetic separator and allow separation to occur for 60 to 120 seconds. 7. With the tube still positioned in the magnetic separator, remove the supernatant. Take care not to disturb the microspheres. 8. Remove the tube from the magnetic separator and resuspend the washed microspheres in 80 ВµL 100 mM Monobasic Sodium Phosphate, pH 6.2 by vortex and sonication for approximately 20 seconds. 9. Add 10 ВµL of 50 mg/mL Sulfo-NHS (diluted in dH20) to the microspheres and mix gently by vortex. 10. Add 10 ВµL of 50 mg/mL EDC (diluted in dH20) to the microspheres and mix gently by vortex. 11. Incubate for 20 minutes at room temperature with gentle mixing by vortex at 10 minute intervals. 12. Place the tube into a magnetic separator and allow separation to occur for 60 to 120 seconds. 13. With the tube still positioned in the magnetic separator, remove the supernatant. Take care not to disturb the microspheres. 14. Remove the tube from the magnetic separator and resuspend the microspheres in 250 ВµL of 50 mM MES, pH 5.0 by vortex and sonication for approximately 20 seconds. See Technical Note 1. 15. Repeat steps 12. and 13. This is a total of two washes with 50 mM MES, pH 5.0. 16. Remove the tube from the magnetic separator and resuspend the activated and washed microspheres in 100 ВµL of 50 mM MES, pH 5.0 by vortex and sonication for approximately 20 seconds. 17. Add 125, 25, 5 or 1 Вµg protein to the resuspended microspheres. (Note: Luminex recommends titration in the 1 to 125 Вµg range to determine the optimal amount of protein per specific coupling reaction.) 3/15/2011 ADV-002 Rev A 7 18. Bring total volume to 500 ВµL with 50 mM MES, pH 5.0. 19. Mix coupling reaction by vortex. 20. Incubate for 2 hours with mixing (by rotation) at room temperature. 21. Place the tube into a magnetic separator and allow separation to occur for 60 to 120 seconds. 22. With the tube still positioned in the magnetic separator, remove the supernatant. Take care not to disturb the microspheres. 23. Remove the tube from the magnetic separator and resuspend the coupled microspheres in 500 ВµL of PBS-TBN by vortex and sonication for approximately 20 seconds. See Technical Note 2. 24. Incubate for 30 minutes with mixing (by rotation) at room temperature. (Note: Optional вЂ“ perform this step when using the microspheres the same day.) 25. Place the tube into a magnetic separator and allow separation to occur for 60 to 120 seconds. 26. With the tube still positioned in the magnetic separator, remove the supernatant. Take care not to disturb the microspheres. 27. Remove the tube from the magnetic separator and resuspend the microspheres in 1 mL of PBS-TBN by vortex and sonication for approximately 20 seconds. See Technical Note 3. 28. Repeat steps 25. and 26. This is a total of two washes with 1 mL PBS-TBN. 29. Remove the tube from the magnetic separator and resuspend the coupled and washed microspheres in 250-1000 ВµL of PBS-TBN. 30. Count the microsphere suspension by hemacytometer, Beckman Coulter Z2в„ў Coulter CounterВ®, Bio-Rad TC10в„ў Automated Cell Counter (see Figure 1), Invitrogen CountessВ® Automated Cell Counter, or Millipore Scepterв„ў cell counter. 31. Calculation on Hemacytometer: Total microspheres = count (1 corner of 4 x 4 section) x (1 x 104) x (dilution factor) x (resuspension volume in mL) 32. Store coupled microspheres refrigerated at 2-8В°C in the dark. 3/15/2011 ADV-002 Rev A 8 Figure 1. Example bead count performed on a Bio-Rad TC10 Cell Counter Technical Note 1: Coupling can be performed in 100 mM MES, pH 6.0 with similar results. For some proteins, better solubility and better coupling may be achieved at a higher coupling pH or in a different buffer. If your protein does not couple satisfactorily under these recommendations, try PBS, pH 7.4 as an alternate coupling buffer. Table 7. Recipe for PBS, pH 7.4 (Alternate Coupling Buffer)*** 1000 mL Reagent PBS, pH 7.4 Catalog Number Sigma P3813 Final Concentration 138 mM NaCl 2.7 mM KCl Amount/ 1L 1 packet Filter Sterilize and store at 4В°C *** An alternative coupling buffer for proteins that do not couple well in 50-100 mM MES, pH 5.0-6.0. Technical Note 2: Either PBS-TBN (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4) or PBS-BN (PBS, 1% BSA, 0.05% Azide, pH 7.4) may be used as Blocking/Storage Buffer. Table 8. Recipe for PBS, 1% BSA, 0.05% Azide, pH 7.4 (PBS-BN Blocking/Storage Buffer) 1000 mL Reagent Catalog Number PBS, 1% BSA, pH 7.4 Sigma P3688 Sodium Azide Filter Sterilize and store at 4В°C Sigma S8032 Final Concentration 138 mM NaCl 2.7 mM KCl 1% BSA 0.05% Azide Amount/ 1L 1 packet 500 mg Technical Note 3: Either PBS-TBN or PBS, 0.05% Tween-20 may be used as Wash Buffer. 3/15/2011 ADV-002 Rev A 9 Confirmation of Coupling Coupling should be confirmed before proceeding to choose the best detection and capture antibodies. Confirmation is key to assay troubleshooting and is a good quality control check for stability over time of your coupled beads. Examples of coupling confirmation can be found in de Jager et al. (2003), Giavedoni (2005), and Lawson et al. (2010). The general protocol for coupling confirmation is to combine your coupled beads and an antispecies IgG (e.g. anti-mouse IgG-PE) that is labeled with phycoerythrin. Typically, a two-fold dilution series starting at 4 Вµg/ml and ending at 0.0625 Вµg/ml is sufficient for coupling confirmation. Table 1 shows a typical assay plate setup for coupling confirmation. Table 9. Example Plate Layout for Coupling Confirmation 1 2 3 Coupled Bead #1 Coupled Bead #2 Coupled Bead #3 PE Anti-Mouse antibody (ug/ml) PE Anti-Mouse antibody (ug/ml) PE Anti-Mouse antibody (ug/ml) A 4 4 4 B 2 2 2 C 1 1 1 D 0.5 0.5 0.5 E 0.25 0.25 0.25 F 0.125 0.125 0.125 G 0.0625 0.0625 0.0625 H 0 0 0 3/15/2011 ADV-002 Rev A 10 What results should be expected from a successful antibody coupling reaction? Antibody coupling is confirmed by testing the coupled microspheres with dilutions of a phycoerythrin-labeled anti-species IgG detection antibody. Typical results are shown below: Figure 2. Coupling confirmation of well and poorly coupled microspheres Coupling Confimation 30000 25000 MFI 20000 15000 10000 5000 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 PE-Antimouse Aby (ug/mL) 3/15/2011 ADV-002 Rev A 11 Figure 3 shows coupling confirmation of an anti-IL-1 antibody coupled at different concentrations. Ideally, you want a curve that saturates somewhere between 10,000 and 20,000 MFI. In this case, you would proceed with either 5 or 25 Вµg per one million beads. Figure 3. Coupling confirmation of Four Different Concentrations of IL-1 Antibody Anti-IL1 Coupling Titration 25000 Median Fluorescence 20000 15000 0.2 ug 1 ug 5 ug 10000 25 ug 5000 0 0 1 2 3 4 5 6 7 8 9 Detection Antibody (ug/mL) 3/15/2011 ADV-002 Rev A 12 In the next case (Figure 4), four different concentrations of an anti-IL-2 antibody coupled to four different bead sets. This is quite different from the previous graph in Figure 3. The three lowest concentrations (1, 5 and 25 Вµg) behave almost identically, so you would proceed with using 1Вµg of anti-IL-2 per one million beads. There is no reason to use more antibody than necessary. Using less capture antibody is one of the most significant cost savings features of using beads over a traditional ELISA. In most situations, 3-10 Вµg per one million beads is optimal, but you should titrate each antibody to determine the optimal concentration. Figure 4. Coupling confirmation of Four Different Concentrations of IL-2 Antibody Anti-IL2 Coupling Titration 25000 20000 0.2ug Median Fluorescence 1 ug 5 ug 15000 25 ug 10000 5000 0 0 1 2 3 4 5 6 7 8 9 Detection Antibody (ug/mL) Things to keep in mind for coupling confirmation: вЂў вЂў вЂў вЂў вЂў вЂў The biggest loss of microspheres occurs from beads sticking to walls of microcentrifuge tubes Use only recommended USA Scientific or Eppendorf Protein low binding tubes Do not remove too much supernatant during wash steps The bead pellet is soft and can be disturbed during supernatant removal Free amines will compete for carboxyl sites on the beads вЂ“ do not use BSA or other proteins in your antibody solution Do not use any buffers containing glycine, TRIS or azide 3/15/2011 ADV-002 Rev A 13 Recommended Protocol for Confirmation of Antibody Coupling Microspheres should be protected from prolonged exposure to light throughout this procedure. Table 10. Required Materials for Antibody Coupling Confirmation Product Antibody-Coupled MagPlex Microspheres Lab-Line Microtiter Plate Shaker Phycoerythrin-Labeled Anti-Species IgG Detection Antibody 96 well microplate aluminum sealing tape (to reduce light exposure of samples in a plate) 96-well round bottom polystyrene solid plates Luminex Magnetic Plate Separator Vortex Mixer Sonicator (mini) Pipettors P10, P20, P100, P1000, 8 ch. MAGPIX Source Catalog Number User Barnstead International Various N/A 4625 Various Costar 6570 Costar 3789 (white) 3792 (black) CN-0269-01 58816-121 08849-00 Various MAGPIX-XPON4.1 Luminex VWR Cole Parmer Rainin or other Luminex Table 11. Recipe for PBS, 1% BSA, pH 7.4 (Assay Buffer) 1000 mL Reagent Catalog Number PBS, 1% BSA, pH 7.4 Sigma P3688 Final Concentration 138 mM NaCl 2.7 mM KCl 1% BSA Amount/ 1L 1 packet Filter Sterilize and store at 4В°C 3/15/2011 ADV-002 Rev A 14 1. Select the appropriate antibody-coupled microsphere sets. 2. Resuspend the microspheres by vortex and sonication for approximately 20 seconds. 3. Prepare a Working Microsphere Mixture by diluting the coupled microsphere stocks to a final concentration of 100 microspheres of each set/ВµL in PBS-1% BSA. (Note: 50 ВµL of Working Microsphere Mixture is required for each reaction.) See Technical Note 1. 4. Prepare two-fold serial dilutions of phycoerythrin-labeled anti-species IgG detection antibody from 4 to 0.0625 Вµg/mL in PBS-1% BSA. (Note: 50 ВµL of diluted detection antibody is required for each reaction.) 5. Aliquot 50 ВµL of the Working Microsphere Mixture into the appropriate wells of the microplate. 6. Add 50 ВµL of the diluted detection antibody into the appropriate wells of the microplate. 7. Mix the reactions gently by pipetting up and down several times with a multi-channel pipettor. 8. Cover the microplate and incubate for 30 minutes at room temperature on a plate shaker. 9. Place the plate into the magnetic separator and allow separation to occur for 60 to 120 seconds. 10. Use a multi-channel pipette to carefully aspirate the supernatant from each well. Take care not to disturb the microspheres. 11. Wash each well twice with 100 ВµL of PBS-1% BSA and aspirate by vacuum manifold. 12. Resuspend the microspheres in 100 ВµL of PBS-1% BSA by gently pipetting up and down five times with a multi-channel pipettor. 13. Analyze 50-75 ВµL on the Luminex analyzer according to the system manual. Technical Note 1: Either PBS-1% BSA or PBS-BN (PBS, 1% BSA, 0.05% Azide, pH 7.4) may be used as Assay Buffer. 3/15/2011 ADV-002 Rev A 15 Selecting the Best Pairs of Antibodies Once all antibodies available are coupled to microspheres, the best pairs should be selected by performing single-plex assays utilizing each combination. Capture and Detection Antibody Selection To perform antibody pair selection, prepare a matrix to determine the best pair. Use one high standard (10,000 pg/ml) as the analyte, as this will allow you to identify the best pairs. Table 12. Example Matrix and Results for Selection of Antibody Pairs Capture Antibodies Antibody A Antibody B Antibody C Detection Antibodies Antibody A Antibody B Antibody C N/A 3583 12321 14832 7920 N/A 8769 N/A 3281 In this example the combination of Antibody C for Detection and Antibody A for capture produced the best results. Assay Protocol/Performing the Assay Start with the recommended protocol below and work from there. Many of the publications in the references section have utilized the standard protocol with good results. In some cases you will need to optimize the protocol. Start with the standard buffers in the protocol. Outline of Capture Sandwich Protocol: 1. Add 50 ul of microspheres to 50 ul of sample 2. Incubation and wash 3. Add 50 ul of detection antibody to antigen bound microspheres 4. Incubation and Wash 5. Add 50 ul of SAPE to detection antibody labeled microspheres 6. Incubation and wash If you are designing a multiplex assay, build each assay individually to roughly optimize as you may need to optimize the multiplex later. Initial rough experiments allow for early assessment of cross reactivity. Cross-reactivity studies are generally performed by running the assay in a multiplex format but with only a single antigen added at high concentration (Bjerre et al. 2009; Joannisson et al. 2006; Kellar et al. 2003). Multiple parameters of the xMAP sandwich immunoassay can be optimized for the critical parameters required for your particular assay. Detection antibody concentration can be titrated for sensitivity (Bjerre et al. 2009). Incubation times can be adjusted to increase sensitivity or reduce background (Bjerre et al. 2009; Dernfalk et al. 2007). In some cases the best sensitivity may be obtained by running more than one smaller multiplex panel instead of one large multiplex 3/15/2011 ADV-002 Rev A 16 (Bjerre et al. 2009). The amount of beads per well can also affect sensitivity. The range should be 2000 to 5000 beads per well. A slight increase in sensitivity can be obtained by using fewer beads, but do not use less than 2000 per well. Pick a buffer for standards that closely mimics the sample composition. Dilute serum or plasma 1:5 (de Jager et al. 2005). Serum and other biological fluids are available from vendors such as Bioreclamation (www.bioreclamation.com). Also, samples should be subjected to only a single freeze-thaw cycle (de Jager et al. 2009), for maximum performance. Aliquot samples if you will analyze over a period of time. Examples of biological fluids used in xMAP assays include tissue culture media (de Jager et al. 2003), serum/plasma (Bjerre et al. 2009; de Jager et al. 2005; Dernfalk et al. 2007; Prabhakar et al. 2002; Ray et al. 2005; Wagner et al. 2009; Wagner et al. 2010), synovial fluid (de Jager et al. 2005), and dried blood spots (Skogstrand et al. 2005). Validation of immunoassays is described in detail in multiple publications (Dernfalk et al. 2007; Bjerre et al. 2009; de Jager et al. 2009; Kellar et al. 2005; Ray et al. 2005; Rizzi et al. 2010; Wood et al. 2011). The parameters selected and optimized will be determined by your particular research needs. The National Institutes of Health Chemical Genomics Center is a good resource for general immunoassay validation (http://www.ncgc.nih.gov/guidance/section10.html). There are also publications that give guidelines for validating assays (Lee et al. 2006; Lee et al. 2009). 3/15/2011 ADV-002 Rev A 17 Recommended Protocol for Washed Capture Sandwich Immunoassay Using Magnetic Microspheres Microspheres should be protected from prolonged exposure to light throughout this procedure. Table 13. Required Materials for a Washed Capture Sandwich Immunoassay Product MagPlex Microspheres coupled with capture antibody Biotinylated Detection antibody Recombinant Protein Standards Streptavidin-R-phycoerythrin (1 mg/ml) OR PhycoLinkВ® Streptavidin-Rphycoerythrin (в‰Ґ1 mg) OR PhycoLinkВ® Streptavidin-Rphycoerythrin (в‰Ґ1 mg) Source User Catalog Number NA Various Various Molecular Probes Prozyme Various Various S-866 PJ31S Prozyme OR Streptavidin-Phycoerythrin Protos Immunoresearch PJRS20 or PJRS14 (next generation of PJ31S) 683 96 well microplate aluminum sealing tape (to reduce light exposure of samples in a plate) 96-well round bottom polystyrene solid plates Luminex Magnetic Plate Separator Costar 6570 Costar Luminex 3789 (white) 3792 (black) CN-0269-01 Lab-Line Microtiter Plate Shaker Barnstead International 4625 Vortex Mixer VWR 58816-121 Sonicator (mini) Cole Parmer 08849-00 Pipettors P10, P20, P100, P1000, 8 ch. Rainin or other Various MAGPIX Luminex MAGPIX-XPON4.1 Table 14. Recipe for PBS, 1% BSA, pH 7.4 (Assay Buffer) 1000 mL Reagent Catalog Number PBS, 1% BSA, pH 7.4 Sigma P3688 Final Concentration 138 mM NaCl 2.7 mM KCl 1% BSA Amount/ 1L 1 packet Filter Sterilize and store at 4В°C 3/15/2011 ADV-002 Rev A 18 Table 15. Recipe for PBS, 0.05% TWEEN-20, pH 7.4 (Wash Buffer) 1000 mL Reagent Catalog Number PBS, 0.05% TWEENВ® 20, pH 7.4 Sigma P3563 Final Concentration 138 mM NaCl 2.7 mM KCl 0.05% TWEEN Amount/ 1L 1 packet Filter Sterilize and store at 4В°C 1. Select the appropriate antibody-coupled microsphere sets. 2. Resuspend the microspheres by vortex and sonication for approximately 20 seconds. 3. Prepare a Working Microsphere Mixture by diluting the coupled microsphere stocks to a final concentration of 100 microspheres of each set/ВµL in PBS-1% BSA. (Note: 50 ВµL of Working Microsphere Mixture is required for each reaction.) See Technical Note 1. 4. Aliquot 50 ВµL of the Working Microsphere Mixture into the appropriate wells of a round-bottom plate. 5. Add 50 ВµL of PBS-1% BSA to each background well. 6. Add 50 ВµL of standard or sample to the appropriate wells. 7. Mix the reactions gently by pipetting up and down several times with a multi-channel pipettor. 8. Cover the plate and incubate for 30 minutes at room temperature on a plate shaker set to approximately 800 rpm. Note: The range for incubation can be from 15 minute to overnight. 9. Place the plate into the magnetic separator and allow separation to occur for 60 to 120 seconds. See Technical Note 2. 10. Use a multi-channel pipette to carefully aspirate the supernatant from each well. Take care not to disturb the microspheres. 11. Leave the plate in the magnetic separator for the following wash steps: a. Add 100 ВµL PBS-1% BSA to each well. b. Use a multi-channel pipette to carefully aspirate the supernatant from each well. Take care not to disturb the microspheres. c. Repeat steps a. and b. above. 12. Remove the plate from the magnetic separator and resuspend the microspheres in 50 ВµL of PBS-1% BSA by gently pipetting up and down several times using a multichannel pipettor. 13. Dilute the biotinylated detection antibody to 4 Вµg/mL in PBS-1% BSA. (Note: 50 ВµL of diluted detection antibody is required for each reaction.) See Technical Note 3. 14. Add 50 ВµL of the diluted detection antibody to each well. 3/15/2011 ADV-002 Rev A 19 15. Mix the reactions gently by pipetting up and down several times with a multi-channel pipettor. 16. Cover the plate and incubate for 30 minutes at room temperature on a plate shaker set to approximately 800 rpm. Note: The range for incubation can be from 15 minutes to several hours). 17. Place the plate into the magnetic separator and allow separation to occur for 60 to 120 seconds. 18. Use a multi-channel pipette to carefully aspirate the supernatant from each well. Take care not to disturb the microspheres. 19. Leave the plate in the magnetic separator for the following wash steps: a. Add 100 ВµL PBS-1% BSA to each well. b. Use a multi-channel pipette to carefully aspirate the supernatant from each well. Take care not to disturb the microspheres. c. Repeat steps a. and b. above. 20. Remove the plate from the magnetic separator and resuspend the microspheres in 50 ВµL of PBS-1% BSA by gently pipetting up and down several times with a multichannel pipettor. 21. Dilute streptavidin-R-phycoerythrin reporter to 4 Вµg/mL in PBS-1% BSA. (Note: 50 ВµL of diluted streptavidin-R-phycoerythrin is required for each reaction.) See Technical Note 3. 22. Add 50 ВµL of the diluted streptavidin-R-phycoerythrin to each well. 23. Mix the reactions gently by pipetting up and down several times with a multi-channel pipettor. 24. Cover the plate and incubate for 30 minutes at room temperature on a plate shaker set to approximately 800 rpm. 25. Place the plate into the magnetic separator and allow separation to occur for 60 to 120 seconds. 26. Use a multi-channel pipette to carefully aspirate the supernatant from each well. Take care not to disturb the microspheres. 27. Leave the plate in the magnetic separator for the following wash steps: a. Add 100 ВµL PBS-1% BSA to each well. b. Use a multi-channel pipette to carefully aspirate the supernatant from each well. Take care not to disturb the microspheres. c. Repeat steps a. and b. above. 28. Remove the plate from the magnetic separator and resuspend the microspheres in 100 ВµL of PBS-1% BSA by gently pipetting up and down several times with a multichannel pipettor. 29. Analyze 50-75 ВµL on the Luminex analyzer according to the system manual. 3/15/2011 ADV-002 Rev A 20 Technical Note 1: Either PBS-1% BSA or PBS-BN (PBS, 1% BSA, 0.05% Azide, pH 7.4) may be used as Assay Buffer. Technical Note 2: For a list of magnetic separator plates, see Recommended Materials for Magnetic Microspheres. Optimal separation time may vary with the type of separator used. Technical Note 3: Concentrations should be optimized for specific reagents, assay conditions, level of multiplexing, etc. in use. Detection antibody concentrations can range from 1-4 Вµg/mL. Streptavidin-PE concentrations can range from 1-4 Вµg/mL. Start with the concentrations in the protocol. 3/15/2011 ADV-002 Rev A 21 References References with Customer-Developed xMAP Assays Bjerre, M., Hansen, T. K., Flyvbjerg, A., and Tonnesen, E. Simultaneous detection of porcine cytokines by multiplex analysis: Development of magnetic bioplex assay. Vet Immunol Immunopathol 2009; 130:53-8. Carson, R. T. and Vignali, D. A. A. Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay. J Immunol Methods 1999; 227:41-52. de Jager, W., te Velthuis, H., Prakken, B. J., Kuis, W., and Rijkers, G. T. Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin Diagn Lab Immunol 2003; 10:133-9. de Jager, W., Prakken, B. J., Bijlsma, J. W. J., Kuis, W., and Rijkers, G. T. Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies. J Immunol Methods 2005; 300:124-35. de Jager, W. and Rijkers, G. T. Solid-phase and bead-based cytokine immunoassay: A comparison. Methods 2006; 38:294-303. de Jager, W., Bourcier, K., Rijkers, G. T., Prakken, B. J., and Seyfert-Margolis, V. Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays. BMC Immunology 2009;10:U1-U11. de Jager W, Prakken B, Rijkers GT. Cytokine Multiplex Immunoassay: Methodology and (Clinical) Applications. T Cell Protocols. 2008:119-33. Dernfalk, J., Waller, K. P., and Johannisson, A. Commercially available antibodies to human tumour necrosis factor-О± tested for cross-reactivity with ovine and bovine tumour necrosis factor-О± using flow cytometric assays. Acta Vet Scand 2004; 45:99-107. Dernfalk, J., Waller, K. P., and Johannisson, A. The xMAPв„ў technique can be used for detection of the inflammatory cytokines IL-1ОІ, IL-6 and TNF-О± bovine samples. Vet Immunol Immunopathol 2007; 118:40-9. Faucher, S., Crawley, A. M., Decker, W., Sherring, A., Bogdanovic, D., Ding, T., Bergeron, M., Angel, J. B., and Sandstrom, P. Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma. PLoS One 2009; 4:U66-U71. Giavedoni, L. D. Simultaneous detection of multiple cytokines and chemokines from nonhuman primates using Luminex technology. J Immunol Methods 2005; 301:89-101. Haasnoot, W. and du Pre, J. G. Luminex-based triplex immunoassay for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powder. J Agric Food Chem 2007; 55:3771-7. 3/15/2011 ADV-002 Rev A 22 Johannisson, A., Jonasson, R., Dernfalk, J., and Jensen-Waern, M. Simultaneous detection of porcine proinflammatory cytokines using multiplex flow cytometry by the xMAPв„ў technology. Cytometry Part A 2006; 69A:391-5. Kellar, K. L., Kalwar, R. R., Dubois, K. A., Crouse, D., Chafin, W. D., and Kane, B.-E. Multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in serum and culture supernatants. Cytometry 2001; 45:27-36. Kellar, K. L. and Douglass, J. P. Multiplexed microsphere-based flow cytometric immunoassays for human cytokines. J Immunol Methods 2003; 279:277-85. Kofoed, K., Schneider, U. V., Scheel, T., Andersen, O., and Eugen-Olsen, J. Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology. Clin Chem 2006; 52:1284-93. Lawson, S., Lunney, J., Zuckermann, F., Osorio, F., Nelson, E., Welbon, C., Clement, T., Fang, Y., Wong, S., Kulas, K., and Christopher-Hennings, J. Development of an 8-plex Luminex assay to detect swine cytokines for vaccine development: Assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination. Vaccine 2010; 28:5356-64. Prabhakar, U., Eirikis, E. and Davis, H. M. Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAPв„ў assay. J Immunol Methods 2002; 260:207-18. Ray, C. A., Bowsher, R. R., Smith, W. C., Devanarayan, V., Willey, M. B., Brandt, J. T., and Dean, R. A. Development, validation, and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum. J Pharm Biomed Anal 2005; 36:1037-44. Rizzi, G., Zhang, Y. J., Latek, R., Weiner, R., and Rhyne, P. W. Characterization and development of a LuminexВ®-based assay for the detection of human IL-23. Bioanalysis 2010; 2:1561-72. Skogstrand, K., Thorsen, P., Norgaard-Pedersen, B., Schendel, D. E., Sorensen, L. C., and Hougaard, D. M. Simultaneous measurement of 25 inflammatory markers and neurotrophinsin neonatal dried blood spots by immunoassay with xMAP technology. Clin Chem 2005; 51:185466. Wagner, B. and Freer, H. Development of a bead-based multiplex assay for simultaneous quantification of cytokines in horses. Vet Immunol Immunopathol 2009; 127:242-8. Wagner, B., Freer, H. and Rollins, A. A fluorescent bead-based multiplex assay for the simultaneous detection of antibodies to B. burgdorferi outer surface proteins in canine serum. Vet Immunol Immunopathol 2011, doi:10.1016/j.vetimm,2010.12.003 Wood, B., OвЂ™Halloran, K. and VandeWoude, S. Development and validation of a multiplex microsphere-based assay for detection of domestic cat (Felis catus) cytokines. Clin Vaccine Immunol 2011, doi:10.1128/CVI.00289-10 3/15/2011 ADV-002 Rev A 23 Assay Validation References Lee, J. W., Devanaryan, V., Barrett, Y. C., Weiner, R., Allinson, J., Fountain, S., Keller, S., Weinryb, I., Green, M., Duan, L., Rogers, J. A., Millham, R., OвЂ™Brien, P. J., Sailstad, J., Khan, M., Ray, C., and Wagner, J. A. Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement. Pharmaceutical Research 2006; 23:312-28. Lee, J. W. and Hall, M. Method validation of protein biomarkers in support of drug development or clinical diagnosis/prognosis. J Chromatogr B Analyt Technol Biomed Life Sci 2009; 877:1259-71. NIH Chemical Genomics Center. Immunoassay Methods. http://www.ncgc.nih.gov/guidance/section10.html ELISA References Engvall, E. The ELISA, Enzyme-Linked Immunosorbent Assay. Clin Chem 2010; 56:319-20. Lequin, R. M. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clin Chem 2005; 51:2415-8. 3/15/2011 ADV-002 Rev A 24 Appendix вЂ“ Automated Washing Option Using Bio-Tek ELx405 Microplate Washer BioTek ELx405 Microplate Washer вЂ“ Keypad Programming Instructions Luminex Mag Bead Assay Wash 96-well Protocol вЂ“ 2 Cycle Wash with No Final Dispense Notes: For use with BioTek-provided magnet P/N 7103016 (Dexter LifeSep 96F technology) and round-bottom 96-well microplate Program delays for 60 seconds with 96-well plate on magnet prior to an aspiration followed by a two cycle wash with 60 second delays after each dispense and ends with a final aspiration Instructions created 1/6/2011 by J. Greene, BioTek Instruments with settings determined by H. Baker, Luminex Corporation 1.) Create SOAK program. DEFINE в†’ CREATE в†’ MORE в†’ SOAK в†’ name (Ex. SOAK60) note: to get letters, scroll through with OPTIONS button в†’ ENTER. SOAK DURATION: 60 SEC в†’ ENTER. SHAKE BEFORE SOAK?: YES в†’ ENTER. SHAKE DURATION: 1 SEC в†’ ENTER. SHAKE INTENSITY: 1 в†’ ENTER. OK TO SAVE PROGRAM? YES в†’ ENTER. 2.) Create ASPIRATION program. DEFINE в†’ CREATE в†’ MORE в†’ ASPIR в†’ name (Ex. ASPIR) note: to get letters, scroll through with OPTIONS button в†’ ENTER. PLATE TYPE: 96 в†’ ENTER. ASPIRATE HEIGHT: 58 в†’ ENTER. HORIZONTAL ASPR POS: -50 в†’ ENTER. HORIZ Y ASPR POS: 0 в†’ ENTER. ASPIRATION RATE: 1 в†’ ENTER. ASPIRATE DELAY: 0 в†’ ENTER. CROSSWISE ASPIR: NO в†’ ENTER. OK TO SAVE PROGRAM? YES в†’ ENTER. 3.) Create WASH program. DEFINE в†’ CREATE в†’ WASH в†’ name (Ex. WASH2X) note: to get letters scroll through with OPTIONS button в†’ ENTER. SELECT REAGENT BOTTLE: A в†’ ENTER. PLATE TYPE: 96 в†’ ENTER. 3/15/2011 ADV-002 Rev A 25 METHOD в†’ NUMBER OF CYCLES: 2 в†’ ENTER в†’ SOAK/SHAKE: YES в†’ ENTER в†’ SOAK DURATION: 60 SEC в†’ ENTER в†’ SHAKE BEFORE SOAK: NO в†’ ENTER в†’ PRIME AFTER SOAK: NO в†’ ENTER. DISP в†’ DISPENSE VOLUME: 100 UL/WELL в†’ ENTER. DISPENSE FLOW RATE: 9 в†’ ENTER. DISPENSE HEIGHT: 128 в†’ ENTER. HORIZONTAL DISP POS: 0 в†’ ENTER. HORIZ Y DISP POS: 0 в†’ ENTER. DISABLE ASPIRATE? NO в†’ ENTER. BOTTOM WASH FIRST?: NO в†’ ENTER. PRIME BEFORE START?: NO в†’ ENTER. ASPIR в†’ ASPIRATE HEIGHT: 58 в†’ ENTER. HORIZONTAL ASPR POS: -50 в†’ ENTER. HORIZ Y ASPR POS: 0 в†’ ENTER. ASPIRATION RATE: 7 в†’ ENTER. ASPIRATE DELAY: 0 в†’ ENTER. CROSSWISE ASPIR: NO в†’ ENTER. FINAL ASPIRATION: YES в†’ ENTER. FINAL ASPIR DELAY: 0 в†’ ENTER. MAIN MENU в†’ OK TO SAVE PROGRAM?: YES в†’ ENTER. 4.) Create LINK program. DEFINE в†’ CREATE в†’ MORE в†’ LINK в†’ name (Ex. LUMINEX) note: to get letters, scroll through with OPTIONS button в†’ ENTER в†’ MORE в†’ SOAK (scroll through using OPTIONS button to find SOAK program you made in step # 1) в†’ ENTER в†’ MORE в†’ ASPIR (scroll through using OPTIONS button to find ASPIR program you made in step # 2) в†’ ENTER в†’ WASH (scroll through using OPTIONS button to find WASH program you made in step # 3) в†’ ENTER. MAIN MENU в†’ OK TO SAVE PROGRAM?: YES в†’ ENTER. 5.) Run LINK program. вЂў вЂў After creating LINK program, this is what you will actually run when washing plates. RUN в†’ MORE в†’ LINK (scroll through with OPTIONS button to find LINK program you made in step # 4) в†’ ENTER. 6.) Basic maintenance. вЂў вЂў 3/15/2011 Before each use: o RUN в†’ PRIME (using your wash buffer) в†’ scroll through using OPTIONS to PRIME 200 в†’ ENTER. End of the day: ADV-002 Rev A 26 o o 3/15/2011 Switch to bottle containing rinse liquid (ex. deionized water). MAINT в†’ scroll through using OPTIONS to OVERNIGHT LOOP в†’ ENTER. ADV-002 Rev A 27